Abstract
Inducible gene expression systems offer researchers the opportunity to synchronize target gene expression at particular developmental stages and in particular tissues. The glucocorticoid receptor (GR), a vertebrate steroid receptor, has been well adopted for this purpose in plants. To generate steroid-inducible plants, a construct of GAL4-binding domain-VP16 activation domain-GR fusion (GVG) with the target gene under the control of upstream activation sequence (UAS) has been developed and extensively used in plant research. Immune receptors perceive conserved molecular patterns secreted by pathogens and initiate robust immune responses. The rice immune receptor, XA21, recognizes a molecular pattern highly conserved in all sequenced genomes of Xanthomonas, and confers robust resistance to X. oryzae pv. oryzae (Xoo). However, identifying genes downstream of XA21 has been hindered because of the restrained lesion and thus limited defense response region in the plants expressing Xa21. Inducible expression allows for a synchronized immune response across a large amount of rice tissue, well suited for studying XA21-mediated immunity by genome-wide approaches such as transcriptomics and proteomics. In this protocol, we describe the use of this GVG system to synchronize Xa21 expression.
Keywords: Dexamethasone, Xa21-mediated immunity, Xanthomonas oryzae pv. oryzae, Glucocorticoid receptor
Materials and Reagents
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Representative data
Figure 1. Experimental design using the dexamethasone-inducible system. Six-week old rice plants are inoculated with Xoo. Before dexamethasone treatment, pTA7002::Myc::XA21 plants (A) do not express XA21 and phenocopy the susceptible Kitaake plants (B). Six days after inoculation, dexamethasone is applied to rice plants and XA21 begins to be expressed, inducing a robust immune response. Leaf tissue is harvested at 0 h, 12 h, and 24 h after dexamethasone application and frozen in liquid nitrogen for downstream applications. Ubi::Myc::XA21 plants(C) overexpressing under the control of the maize ubiquitin promoter (Park et al., 2010) are resistant to Xoo and used as a positive control here. Figure 2. Expression of Xa21 after dexamethasone application. pTA7002::Myc::XA21 rice leaves were harvested at the indicated time points after application of dexamethasone. RNA was extracted using TRIzol with standard protocol; cDNA used for quantitative PCR was transcribed with M-MLV reverse transcriptase; and quantitative PCR was performed with SsoFastEvaGreenSupermix on a PCR machine. The gene expression of Xa21 was normalized using the rice ubiquitin gene (LOC_Os06g46770) as an internal control, and the expression level in the Ubi::Myc::XA21 plants (labeled as XA21) was set as 1.0. Figure 3. Dexamethasone treatment inhibits the elongation of lesion lengths in pTA7002::Myc::XA21 plants. Six week old rice plants were inoculated with Xoo. At six days after inoculation (6 DAI) disease progression was measured (red line) before dexamethasone (DEX) was applied to the plants. Until 6 DAI, pTA7002::Myc::XA21 plants did not express Xa21 and the observed variation in disease development between with DEX and without DEX treated plants was not statistically significant. Disease progression was allowed to continue until 12 days after inoculation (12 DAI, 6 days after the DEX treatment), and lesion length was measured as indicated (blue line). pTA7002::Myc::XA21 plants treated with dexamethasone showed significantly less disease progression compared to non-dexamethasone treated plants indicating that DEX treatment was successful in inducing expression of Xa21 and that XA21-mediated immunity was triggered.
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Acknowledgments
This protocol was modified from Park et al. (2012). This research was supported by the National Institute of Health (NIH, GM55962), the National Science Foundation (NSF, IOS-0817738), and the Monsanto's Beachell-Borlaug International Scholars Program.
References
Appendix
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